Method for determining the immunogenicity of an antigen

ABSTRACT

The present invention relates to methods for preparing an artificial immune system. The artificial immune system comprises a cell culture comprising T cells, B cells and antigen-primed dendritic cells. The artificial immune system of the present invention can be used for in vitro testing of vaccines, adjuvants, immunotherapy candidates, cosmetics, drugs, biologics and other chemicals.

CROSS REFERENCE TO RELATED CASES

This application is a divisional of U.S. application Ser. No.11/594,172, filed Nov. 8, 2006, which is continuation-in-part of U.S.application Ser. No. 11/453,046, filed Jun. 15, 2006, which is acontinuation-in-part of U.S. application Ser. No. 11/116,234, filed Apr.28, 2005, which claims the benefit of U.S. Provisional Application Ser.Nos. 60/565,846, filed Apr. 28, 2004, and 60/643,175, filed Jan. 13,2005. This application also claims the benefit of priority ofInternational Application No. PCT/US05/014444, filed Apr. 28, 2005. Eachof these applications is hereby incorporated by reference in theirentirety

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under contract numberNBCHC060058, awarded by the Defense Advanced Research Projects Agency,issued by the U.S. Army Medical Research Acquisition Activity, andadministered by the U.S. Department of the Interior-National BusinessCenter. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to a method for constructing anintegrated artificial human tissue construct system and, in particular,construction of an integrated human immune system for in vitro testingof vaccines, adjuvants, immunotherapy candidates, cosmetics, drugs,biologics, and other chemicals. The artificial immune system of thepresent invention is useful for assessing the interaction of substanceswith the immune system, and thus can be used to accelerate and improvethe accuracy and predictability of, for example, vaccine, drug,biologic, immunotherapy, cosmetic, and chemical development.

2. Background of the Technology

Despite the advent and promise of recent technologies, includingcombinatorial chemistry, high-throughput screening, genomics, andproteomics, the number of new drugs and vaccines reaching the market hasnot increased. In fact, the attrition rate within drug discoveryprograms exceeds 90%.

The introduction of these new (and expensive) technologies has notreduced the lost opportunity costs associated with immunotherapydevelopment; rather, these costs have increased. Indeed, it is nowestimated that almost $1 billion is required to bring a new drug to themarket.

The development and biological testing of human vaccines hastraditionally relied on small animal models (e.g., mouse and rabbitmodels) and then non-human primate models. However, such small animalmodels are expensive and non-human primate models are both expensive andprecious. Furthermore, there are many issues regarding the value of suchanimal studies in predicting outcomes in human studies.

A major problem remains the translation from test systems to humanimmunology. Successful transfer between traditional testing systems andhuman biology requires an intricate understanding of diseasepathogenesis and immunological responses at all levels. Given worldwidehealth problems caused by known and emerging infectious agents and evenpotential biological warfare pathogens, it is time for a fresh approachto understanding disease pathogenesis, the development and rapid testingof vaccines, and insights gathered from such work.

The body's distributed immune system can be roughly divided into fourdistinct compartments: tissues and blood, mucosal tissues, bodycavities, and skin. Because of ease of study, most is known about thetissue and blood compartment and its lymphoid tissues, the spleen andlymph nodes.

The mammalian immune system uses two general adaptive mechanisms toprotect the body against environmental pathogens. When apathogen-derived molecule is encountered, the immune response becomesactivated to ensure protection against that pathogenic organism.

The first immune system mechanism is the non-specific (or innate)inflammatory response. The innate immune system appears to recognizespecific molecules that are present on pathogens but not within the bodyitself.

The second immune system mechanism is the specific or acquired (oradaptive) immune response. Innate responses are fundamentally the samefor each injury or infection; in contrast, acquired responses arecustom-tailored to the pathogen in question. The acquired immune systemevolves a specific immunoglobulin (antibody) response to many differentmolecules, or antigens, derived from the pathogen. In addition, a largerepertoire of T cell receptors (TCR) is sampled for their ability tobind processed peptides from the antigens that are bound by majorhistocompatibility complex (MHC) class I and II proteins on the surfaceof antigen-presenting cells (APCs), such as dendritic cells (DCs).

Acquired immunity is mediated by specialized immune cells called B and Tlymphocytes (or simply B and T cells). Acquired immunity has specificmemory for specific antigens; repeated exposure to the same antigenincreases the memory response, which increases the level of inducedprotection against that particular pathogen.

B cells produce and mediate their functions through the actions ofantibodies. B cell-dependent immune responses are referred to as“humoral immunity” because antibodies are found in body fluids.

T cell-dependent immune responses are referred to as “cell-mediatedimmunity,” because effector activities are mediated directly by thelocal actions of effector T cells. The local actions of effector T cellsare amplified through synergistic interactions between T cells andsecondary effector cells, such as activated macrophages. The result isthat the pathogen is killed and prevented from causing diseases.

The functional element of a mammalian lymph node is the follicle, whichdevelops a germinal center (GC) when stimulated by an antigen. The GC isan active area within a lymph node, where important interactions occurin the development of an effective humoral immune response. Upon antigenstimulation, follicles are replicated and an active human lymph node mayhave dozens of active follicles, with functioning GCs. Interactionsbetween B cells, T cells, and FDCs take place in GCs.

Various studies of GCs in vivo indicate that the many important eventsoccur there, including immunoglobulin (Ig) class switching, rapid B cellproliferation (GC dark zone), production of B memory cells, accumulationof select populations of antigen-specific T cells and B cells,hypermutation, selection of somatically mutated B cells with highaffinity receptors, apoptosis of low affinity B cells, affinitymaturation, induction of secondary antibody responses, and regulation ofserum immunoglobulin G (IgG) with high affinity antibodies. Similarly,data from in vitro GC models indicate that FDCs are involved instimulating B cell proliferation with mitogens and it can also bedemonstrated with antigen (Ag), promoting production of antibodiesincluding recall antibody responses, producing chemokines that attract Bcells and certain populations of T cells, and blocking apoptosis of Bcells.

Similar to pathogens, vaccines function by initiating an innate immuneresponse at the vaccination site and activating antigen-specific T and Bcells that can give rise to long term memory cells in secondary lymphoidtissues. The precise interactions of the vaccine with cells at thevaccination site and with T and B cells of the lymphoid tissues areimportant to the ultimate success of the vaccine.

Almost all vaccines to infectious organisms were and continue to bedeveloped through the classical approach of generating an attenuated orinactivated pathogen as the vaccine itself. This approach, however,fails to take advantage of the recent explosion in our mechanisticunderstanding of immunity. Rather, it remains an empirical approach thatconsists of making variants of the pathogen and testing them forefficacy in non-human animal models.

Advances in the design, creation and testing of more sophisticatedvaccines have been stalled for several reasons. First, only a smallnumber of vaccines can be tested in humans, because, understandably,there is little societal tolerance for harmful side effects in healthypeople, especially children, exposed to experimental vaccines. With theexception of cancer vaccine trials, this greatly limits the innovationthat can be allowed in the real world of human clinical trials. Second,it remains challenging to predict which immunodominant epitopes areoptimal for induction of effective CD4⁺ and CD8⁺ T cell responses andneutralizing B cell responses. Third, small animal testing, followed byprimate trials, has been the mainstay of vaccine development; suchapproaches are limited by intrinsic differences between human andnon-human species, and ethical and cost considerations that restrict theuse of non-human primates. Consequently, there has been a slowtranslation of basic knowledge to the clinic, but equally important, aslow advance in the understanding of human immunity in vivo.

The artificial immune system (AIS) of the present invention can be usedto address this inability to test many novel vaccines in human trials byinstead using human tissues and cells in vitro. The AIS enables rapidvaccine assessment in an in vitro model of human immunity. The AISprovides an additional model for testing vaccines in addition to thecurrently used animal models.

Attempts have been made in modulating the immune system. See, forexample, U.S. Pat. No. 6,835,550 B1, U.S. Pat. No. 5,008,116, WO2004/101773 A1, Suematsu et al., (Nat Biotechnol, 22, 1539-1545, (2004))and U.S. Patent Application No. 2003/0109042.

Nevertheless, none of these publications describe or suggest anartificial (ex vivo) human cell-based, immune-responsive systemcomprising a vaccination site (VS) and a lymphoid tissue equivalent(LTE). The present invention comprises such a system and its use inassessing the interaction of substances with the immune system.

SUMMARY OF THE INVENTION

The present invention is directed to artificial immune systemscomprising cell cultures of B cells, T cells and antigen-primeddendritic cells.

The present invention is also directed to methods for detecting animmune response to an antigen using the cell cultures of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows the detection of tetanus-specific antibody responses byELISPOT and determination of the percentage of antigen-specific B cellsusing a 2D T and B cell co-culture.

FIG. 2: Depicts tetanus toxoid: B cell proliferation and comparisonbetween PBMC and 2D T and B cell co-culture.

FIG. 3: Shows the flow cytometry data indicating B cell proliferationbetween PBMC and 2D T and B cell co-culture for the same cell donorshown in FIG. 2.

FIG. 4: Depicts tetanus toxoid-specific ELISPOT comparing PBMC to 2D Tand B cell co-culture for the same cell donor shown in FIGS. 2 and 3.

FIG. 5: Shows an in vitro system representative of the physiologicalstate promotes stronger B cell proliferative and tetanus toxoid-specificantibody responses, using a 2D co-culture of T and B cells and TT-pulsedDCs.

FIG. 6: Depicts tetanus-specific antibody responses to a DTaP(diphtheria and tetanus and acellular pertussis vaccine, adsorbed)vaccine and a simple tetanus toxoid Antigen, using a 2D co-culture of Tand B cells and TT-pulsed DCs.

FIG. 7: Shows the influence of vaccine versus antigen in a lymphoidtissue equivalent (LTE) for the same cell donor shown in FIG. 6.

FIG. 8: Depicts Strong B cell and T cell proliferative responses seenagainst C. albicans, associated with potent activation (HLA-DR^(high),CD86^(high)) of the dividing B cells using a 2D co-culture of T and Bcells and TT-pulsed DCs.

FIG. 9: Shows specificity of the C. albican-stimulated B cellsdemonstrated by ELIPSOT for the same donor in FIG. 8. C.albicans-specific ELISPOT data comparing compares the 2D co-culture of Tand B cells with PBMCs.

FIG. 10: Depicts antibody responses when some of the leukocytes areremoved.

FIG. 11: Shows in vitro antigen-specific antibody response to influenza.

FIG. 12: Shows T and B cell proliferation induced by H1N1 influenza.

FIG. 13: Shows in vitro the importance of the temporal sequence ofimmunological events.

FIG. 14: Shows a rapid assay system for the development of primary Bcell responses.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns the development of accurate, predictivein vitro models to accelerate vaccine testing, allow collection of moreinformative data that will aid in redesigning and optimizing vaccineformulations before animal or clinical trials, and raise the probabilitythat a vaccine candidate will be successful in human trials. Morespecifically, the present invention comprises controlling the nature andstate of the cells in the lymphoid tissue equivalent (LTE, artificiallymph node) of the artificial immune system (AIS).

The AIS can be used to test vaccines and other pharmaceuticals forimmune reactivity in a manner that is more predictive than animalexperiments. Consequently, it can provide valuable pre-clinical dataearlier in the research and development process. Antigenic moleculesintroduced to the AIS are acquired by dendritic cells (DCs) at thevaccination site (VS). The DCs are then transferred to the lymphoidtissue equivalent (LTE), where they present the antigen to T cells,activating their immune function. Activated helper T cells co-stimulateB cells to induce antibody production, while activated cytotoxic T cellslyse antigen-bearing cells. Solubilized antigen(s) can also beintroduced into the LTE to directly activate B cells for subsequentantibody production.

While a number of published reports have demonstrated antigen-specific Bcell responses (to C. albicans, TT, and other antigens) in vitro, theseresults are typically achieved by stimulating and restimulating culturesof whole PBMCs with antigen and exogenous factors to boost B cellproliferation and/or activation.

The present invention comprises the detection of antibody responsesusing defined cultures of B cells, T cells, and DCs and optionallyfollicular dendritic cells (FDCs), in 2-dimensional construct assay. Thepresence of secondary cells provides a more physiological environmentfor B cell activation and differentiation, such that artificial factorsin the cultures are not necessary to detect specific antibody responses.In an embodiment of the present invention, the LTE comprises allogeneicT cells. In another embodiment, the LTE comprises autologous T cells.

Using embodiments of the present invention, we have generatedantigen-specific B cell responses using a 2-dimensional (2D) co-culturesystem comprising T cells, B cells, and antigen-pulsed DCs. In theexamples, responses were generated against tetanus toxoid (TT) and awhole protein extract of Candida albicans (C. albicans). The resultsfrom these examples show that culturing human T and B cells together invitro at a ˜1:1 ratio, versus the ratio of T and B cells naturally foundin the blood, gave stronger antigen responses, by both analysis ofactivation and proliferation (flow cytometry) and antibody production(ELISPOT). Although the preferred ratio of T cells:B cells is ˜1:1, theratio of T cells:B cells can range from ˜1:10 to ˜10:1. In the culturesof the examples, “T cells” included both CD4⁺ and CD8⁺ T cells. Inperipheral blood, the T (total T cells):B cell ratio is ˜7:1. In thelymph node, the T (total T cells):B cell ratio is ˜1:1.6. In thegerminal center, the T cell:B cell ratio is ˜1:8, and there, the T cellsare primarily CD4⁺ T cells.

In the results of the experiments shown, engineered serum-free media(X-VIVO) was used, though we have also used serum (e.g., human, bovine)in other experiments (data not shown). Dendritic cells (DCs) weregenerated from CD14-purified monocytes that were cultured for ˜7 days inX-VIVO 15 media, supplemented with GM-CSF (˜100 ng/ml) and IL-4 (˜25ng/ml). The cytokine-derived DCs were pulsed with antigen or vaccine andthen cocultured with T cells. After adding the antigen-prepulseddendritic cells to the T cell culture, B cells primed with the sameantigen used to prime the DCs are added to the cell culture. Afteradding the antigen-primed B cells to the cell culture, further solubleantigen can also be added. For PBMC cultures, either the antigen wasadded to the assay, or antigen-pulsed DCs were added to the assay. InFIGS. 1 to 9, antigen-pulsed DCs were added to the co-culture of T and Bcells, while soluble antigen was added to the PBMC cultures. FIG. 9shows a comparison of the co-culture to PBMCs, with antigen-pulsed DCsadded to both systems.

Antibodies specific for the antigen of interest can be isolated from theresulting cell cultures of the present invention. Such antibodies can beused for a variety of purposes, including in therapeutic and diagnosticmethods.

In addition, antigen-specific B cells can be isolated, cloned andimmortalized from the cell cultures of the present invention, and canalso be used in therapeutic and diagnostic methods.

Alternatively, all of the antibody-producing B cells are collected enmasse (without isolating/cloning individual B cells) for immortalizingand producing a therapeutic.

Thus, the present invention also encompasses methods of producing atherapeutic comprising antibodies or antibody-producing B cells incombination with a pharmaceutically acceptable carrier.

EXAMPLES

These experiments provide a direct comparison of PBMCs versus aco-culture of negatively selected T and B cells that were plated at a˜1:1 ratio in—in these examples—a 96-well, round bottom plate. Allassays were harvested on day 7 of in vitro culture. All experiments wereanalyzed by ELISPOT for antibody production and by flow cytometry forproliferation, as determined by loss of CFSE. In the ELISPOT assaysbecause there were different ratios of T and B cells in the PBMC culturecompared with the TB-2D cultures, there were fewer B cells plated intothe ELISPOT wells. However, in the experiment in FIG. 4, the numbers ofB cells used in the ELISPOT experiments for both the PBMC and co-cultureassays were approximately equal. We determined the approximate number ofB cells in the ELISPOT wells by flow cytometry to enable comparisons.

These results show that culturing human T and B cells together in vitroat a ˜1:1 ratio compared to the ratio of T and B cells naturally foundin the blood give stronger antigen responses, by both analysis ofactivation and proliferation (flow cytometry) and antibody production(ELISPOT). The results also show that co-culturing T cells andantigen-primed dendritic cells, and subsequently adding antigen-primed Bcells, also gives stronger antigen responses, as indicated by antibodyproduction (ELISPOT).

Example 1

B and T cell co-culture with tetanus toxoid, showing the ability todetect tetanus-specific antibody responses.

Example 2a

PBMC versus co-culture, using a tetanus toxoid antigen. Even thoughsimilar B cell proliferation responses were seen in PBMC and 2D T and Bcell co-cultures (FIGS. 2, 3), an improved tetanus toxoid-specificantibody response was observed in a T and B cell co-culture LTE, ascompared with PBMC cultures (FIG. 4).

Example 2b

PBMC versus co-culture, using Candida albicans antigens. FIG. 9 shows C.albicans-specific ELISPOT data, comparing TB-2D to PBMCs. In thisexperiment, DCs were pulsed with TT antigen only, but the ELISPOT wasconducted on both TT- and C. albicans-coated plates.

Example 2c

PBMC versus co-culture (FIG. 10). In this example we addressed thequestion of what happens if we take cells from an apparent“non-responder” and use only the GC cells from the leukocytes. Note theresponse when some of the leukocytes are removed (FIG. 10);non-responders in vitro now show an antibody response.

Here, we used human CD4⁺ T and B cells with FDCs and formed GCs in vitroand then examined whether IgG production could be obtained against arecall antigen. Specifically, we used tetanus toxoid (TT) in theseexperiments and isolated human B cells and CD4⁺ T cells from peripheralblood.

We observed IgG recall responses using only the T cells, B cells, andFDCs that are typically found in GCs. In contrast, in the presence ofPBL cells not normally in found in GCs, no antibody response wasdetectible in cells from some donors. These results show that removing(not including) other cells, such NK cells, monocytes, and CD8⁺ T cells,improved the IgG response.

Example 3

In vitro system representative of the physiological state promoteshigher B cell proliferative and tetanus toxoid-specific antibodyresponses following tetanus vaccination (FIG. 5). The post tetanustoxoid experiment was conducted 5 weeks following vaccination. Thetetanus antibody titer before vaccination was ˜40 μg/mL; aftervaccination it was ˜300 μg/mL. T cells represent both CD4⁺ and CD8⁺ Tcells. Peripheral blood has a T:B ratio of ˜7:1 (total T cells). Thelymph node has a T:B ratio of ˜1:1.6 (total T cells). The germinalcenter has a T:B ratio of ˜1:8 (primarily CD4⁺ T cells).

Example 4

Use of a vaccine to elicit in vitro immune responses in a co-culture ofT and B cells. DCs were pulsed with the vaccine or the tetanus toxoidantigen and were then added to the co-culture of T and B cells.Tripedia® (diphtheria and tetanus toxoids and acellular pertussisvaccine, adsorbed; DTaP), for intramuscular use, is a sterilepreparation of diphtheria and tetanus toxoids adsorbed, with acellularpertussis vaccine in an isotonic sodium chloride solution containingthimerosal (preservative) and sodium phosphate (to control pH). Aftershaking, the vaccine is a homogeneous white suspension. Tripedia®vaccine is distributed by Aventis Pasteur Inc.

Example 5

To detect antigen-specific antibody responses, we developed an ELISPOTapproach to quantify B cell responses (antigen specificity) on a percell basis. In this example, T cells were cultured with B cells at a˜1:1 ratio, with cytokine-derived DCs included at a DC:T and B (total)cell ratio of ˜1:60. Soluble TT (˜1 μg/ml) or C. albicans (˜10 μg/ml)was included for the entire 7-day culture, while other wells receivedpokeweed mitogen (PWM; a strong, non-specific lymphocyte stimulator) forthe final 3 days of the culture.

On the seventh day, the lymphocytes were examined for marker expressionand CFSE profiles by flow cytometry and the frequency of TT and C.albican-specific B cells was calculated by ELISPOT. Briefly, ˜30×10³total lymphocytes were plated in duplicate wells of an ELISPOT platethat had been pre-coated with TT, C. albicans, or anti-immunoglobulin(Ig, to gauge total antibody production).

The cells were then serially diluted five times at a ˜1:3 ratio and PWMwas added to all wells to trigger antibody production. The cells werethen incubated for ˜5 hr at 37° C. in a 5% CO₂ incubator and washedaway. Plate-bound antibody was detected using techniques similar tothose required for ELISA.

The results in FIG. 8 demonstrate strong B cell and T cell proliferativeresponses against C. albicans, associated with potent activation(HLA-DR^(high), CD86^(high)) of the dividing B cells. Furthermore, asubset of the most divided B cells appears to have acquired a memoryphenotype, indicated by increased CD27 expression.

The lack of a robust response against TT was consistent with the weakserum TT titer for this donor (˜4 μg/ml). As expected, PWM triggeredpotent T and B cell proliferative responses, though not as manydivisions were seen as with specific antigen stimulation, likely becausethe cells were only cultured with the mitogen for 3 days.

The specificity of the C. albicans-stimulated B cells was demonstratedby ELIPSOT (FIG. 2). This experiment suggests that a 1× stimulation withC. albicans did give rise to a small population of antibody-producingcells (˜0.2% of total B cells) that was not detected in untreatedcultures or those stimulated with TT (left and middle wells). Thisdiscrepancy between the frequency of proliferating cells and C.albicans-specific B cells detected by ELISPOT could be the result ofseveral factors. A likely explanation is that we used a crude C.albicans whole antigen extract containing ˜19% carbohydrates (byweight). While C. albicans polysaccharides are strong inducers of B cellresponses, only protein antigen-specific responses would be detected inthe ELISPOT assay.

Example 6

Tetanus-specific antibodies were detected in another ELISPOT experimentwhere the cell donor's serum anti-tetanus level was higher (63 μg/ml),and DCs were cultivated in XVIVO-15 medium. All other components,concentrations and ratios were left unchanged, except that of the numberof cells deposited per ELISPOT well was increased; the higher numberused was ˜1×10⁵ cells/well.

In this experiment, both TT- and C. albicans-specific antibodies wereobserved (up to 48 and 33 spots per well, respectively), although a highlevel of non-specific response, especially in the presence ofCCL21/anti-CD40 additives, did not allow a firm conclusion in favor ofantigen-specific versus mitogenic activity.

Example 7

The specificity of the C. albicans-stimulated B cells was demonstratedby ELIPSOT (FIG. 9) for both PBMC and 2D co-culture of T and B cellswith C. albicans-pulsed DCs added to both systems. This experimentindicates that even if the PBMC cultures have antigen-pulsed DCs addedthat the co-culture system shows a stronger antibody response, asdetermined by ELISPOT.

Example 8

In vitro antigen-specific antibody response to influenza (FIG. 11) and Tand B cell proliferation induced by H1N1 influenza (FIG. 12). DCs weretreated (or not) with H1N1 (New Caledonia) influenza. 2D cultures of DCsand T and B cells were stimulated (or not) with ‘soluble’ H1N1influenza. As can be seen, there was antigen-specific proliferation of Tand B lymphocytes and generation of antigen-specific antibody secretingB lymphocytes (ELISPOT data). Note the largest (apparently synergistic)response was observed when we pulsed the DCs with antigen and then addedsoluble antigen to the DC/T and B cell cultures, to activate the Bcells, which are antigen-presenting cells (APCs). Again, the T and Bcell co-culture is superior to PBMC cultures.

Example 9

In this example, the antigens examined were tetanus toxoid (TT) and awhole protein extract of Candida albicans.

CD4⁺ T cells, purified by negative magnetic bead selection, werecultured with antigen-pulsed DCs at ˜1:60 ratio.

In the first experiment, B cells were added at the same time (day 0) inequal numbers to the T cells, DCs and antigen.

In the second experiment, antigen-primed B cells were added to the T/DCcultures 3 days after the T/DC cultures were established.

As a control, cultures were also established in the absence of anyantigen.

Seven days following the addition of the antigen-primed B cells to theT/DC cultures, the cells were harvested and analyzed forantigen-specific B cells by ELISPOT assay.

As FIG. 13 illustrates, the immune (antibody) response was much strongerin the case where the antigen-primed B cells were added to the T/DCculture after 3 days. Similar results were obtained using cells obtainedform three independent blood donors.

Example 10

A rapid assay system for the development of primary B cell responses.Antigen-primed dendritic cells (DCs) were cultured with syngeneic Bcells (˜2 to ˜2.5×10⁶) in 24-well plates and allogeneic CD4⁺ T cells ata 1:100 ratio (i.e., ˜2 to ˜2.5×10⁴ allogeneic T cells). Additionalantigen was added (the antigens used were keyhole limpet hemocyanin(KLH) and the anthrax recombinant protective antigen (rPa)). Fourteendays later, the primary B cell response was assessed by ELISPOT andFACS. As FIG. 14 illustrates, the ELISPOT results show that, despitesome background, enhanced primary B cell responses (IgM) were observedto the antigens.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be appreciated by one skilled in the art from reading thisdisclosure that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

1. A method for immortalizing B cells monoclonal for an antigen,comprising: (a) priming a population of dendritic cells with an antigen;(b) adding the antigen-primed dendritic cells of (a) to a cell culturecomprising T cells and serum-free culture media; (c) priming apopulation of B cells with the antigen; (d) adding the antigen-primed Bcells of (c) to the cell culture comprising T cells and antigen-primeddendritic cells of (b); (e) culturing the cell culture of (d) underconditions promoting production of antibodies by the antigen-primed Bcells; (f) identifying B cells in the culture of (e) that are monoclonalfor the antigen; and (g) isolating and immortalizing B cells identifiedin (f) that are monoclonal for the antigen.
 2. The method of claim 1,wherein the serum-free cell culture media is X-VIVO
 15. 3. The method ofclaim 1, further comprising adding the antigen to the cell culture (e).4. A method for producing antibodies having binding specificity for anantigen, comprising: (a) priming a population of dendritic cells with anantigen; (b) adding the antigen-primed dendritic cells of (a) to a cellculture comprising T cells and serum-free culture media; (c) priming apopulation of B cells with the antigen; (d) adding the antigen-primed Bcells of (c) to the cell culture comprising T cells and antigen-primeddendritic cells of (b); and (e) culturing the cell culture of (d) underconditions promoting production of antibodies by the antigen-primed Bcells.
 5. The method of claim 4, further comprising isolating antibodiesspecific for the antigen from the culture of (e).
 6. The method of claim4, wherein the serum-free cell culture media is X-VIVO
 15. 7. The methodof claim 4, further comprising adding the antigen to the cell culture(e).
 8. A method for immortalizing B cells producing antibodies havingbinding specificity for an antigen, comprising: (a) priming a populationof dendritic cells with an antigen; (b) adding the antigen-primeddendritic cells of (a) to a cell culture comprising T cells andserum-free cell culture media; (c) priming a population of B cells withthe antigen; (d) adding the antigen-primed B cells of (c) to the cellculture comprising T cells and antigen-primed dendritic cells of (b);(e) culturing the cell culture of (d) under conditions promotingproduction of antibodies by the antigen-primed B cells; (f) identifyingB cells producing antibodies having binding specificity for the antigenin the culture of (e); and (g) isolating and immortalizing B cellsidentified in (f) producing antibodies having binding specificity forthe antigen.
 9. The method of claim 8, wherein the serum-free cellculture media is X-VIVO
 15. 10. The method of claim 8, furthercomprising adding the antigen to the cell culture of (e).
 11. A methodfor producing a therapeutic composition comprising monoclonal antibodieshaving binding specificity for an antigen, the method comprising: (a)priming a population of dendritic cells with an antigen; (b) adding theantigen-primed dendritic cells of (a) to a cell culture comprising Tcells and serum-free cell culture media; (c) priming a population of Bcells with the antigen; (d) adding the antigen-primed B cells of (c) tothe cell culture comprising T cells and antigen-primed dendritic cellsof (b); (e) culturing the cell culture of (d) under conditions promotingproduction of antibodies by the antigen-primed B cells; (f) identifyingB cells in the culture of (e) that are monoclonal for the antigen; (g)isolating and immortalizing B cells identified in (f) that aremonoclonal for the antigen; and (h) preparing a therapeutic compositioncomprising monoclonal antibodies produced by the immortalized B cells of(g) and a pharmaceutically acceptable carrier.
 12. A method forproducing a therapeutic composition comprising a monoclonal antibodyhaving binding specificity for an antigen, the method comprising: (a)priming a population of dendritic cells with an antigen; (b) adding theantigen-primed dendritic cells of (a) to a cell culture comprising Tcells and serum-free cell culture media; (c) priming a population of Bcells with the antigen; (d) adding the antigen-primed B cells of (c) tothe cell culture comprising T cells and antigen-primed dendritic cellsof (b); (e) culturing the cell culture of (d) under conditions promotingproduction of antibodies by the antigen-primed B cells; (f) identifyingB cells in the culture of (e) that are monoclonal for the antigen; (g)isolating and immortalizing B cells identified in (f) that aremonoclonal for the antigen; and (h) preparing a therapeutic compositioncomprising monoclonal antibodies produced by one of the immortalized Bcells of (g) and a pharmaceutically acceptable carrier.
 13. A method forproducing a therapeutic composition comprising antibodies having bindingspecificity for an antigen, the method comprising: (a) priming apopulation of dendritic cells with an antigen; (b) adding theantigen-primed dendritic cells of (a) to a cell culture comprising Tcells and serum-free cell culture media; (c) priming a population of Bcells with the antigen; (d) adding the antigen-primed B cells of (c) tothe cell culture comprising T cells and antigen-primed dendritic cellsof (b); (e) culturing the cell culture of (d) under conditions promotingproduction of antibodies by the antigen-primed B cells; (f) identifyingB cells in the culture of (e) producing antibodies having bindingspecificity for the antigen; (g) isolating and immortalizing B cellsidentified in (f) having binding specificity for the antigen; and (h)preparing a therapeutic composition comprising antibodies having bindingspecificity for the antigen produced by the immortalized B cells of (g)and a pharmaceutically acceptable carrier.
 14. A method for preparing atherapeutic composition comprising antibodies having binding specificityfor an antigen, the method comprising: (a) priming a population ofdendritic cells with an antigen; (b) adding the antigen-primed dendriticcells of (a) to a cell culture comprising T cells and serum-free cellculture media; (c) priming a population of B cells with the antigen; (d)adding the antigen-primed B cells of (c) to the cell culture comprisingT cells and antigen-primed dendritic cells of (b); (e) culturing thecell culture of (d) under conditions promoting production of antibodiesby the antigen-primed B cells; (f) isolating antibodies specific for theantigen from the culture of (e); and (g) preparing a therapeuticcomposition comprising the isolated antibodies of (f) and apharmaceutically acceptable carrier.